Site-directed mutant p21 proteins defective in both inhibition of E2F-regulated transcription and disruption of E2F-p130-cyclin-cdk2 complexes.
P21 is a regulatory protein that can contribute to cell cycle arrest by inhibiting the cyclin-dependent-kinases (cdks). However, the mechanism that links the inhibition of the cdk activities and the cell cycle arrest is not well established. To investigate this, we studied a purified endogenous cellular complex which contained E2F (in the form of E2F-4), p130, cyclin, and cdk2. This complex of E2F-p130-cyclin-cdk2 is found mainly in cycling cells and is postulated to be an intermediate that leads to the activation of E2F. We previously showed that p21 could disrupt this complex leading to the accumulation of an E2F-p130 complex and the inhibition of E2F-regulated transcription. We analyzed a group of p21 mutants including those that harbored changes in cyclin- and cdk2-binding motifs. We show that both the cyclin and cdk2 binding motifs of p21 are crucial for the disruption of this endogenous complex of E2F-p130-cyclin-cdk2. This suggests a model where the ability of p21 to inhibit the function of this complex is dependent on interactions with both cyclin and cdk2 molecules. This was substantiated by studies with intact cells. P21 mutants that are impaired in their ability to disrupt the cellular E2F-p130-cyclin-cdk2 complex are also shown to be maximally impaired in the ability to repress E2F-regulated transcription.
Robles
S J
SJ
Department of Oral Medicine and Diagnostic Sciences, College of Dentistry, University of Illinois at Chicago, 60612, USA.
Shiyanov
P
P
Aristodemo
G T
GT
Raychaudhuri
P
P
Adami
G R
GR
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D016213:Cyclins / Q000502:physiology*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050704:E2F4 Transcription Factor; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000502:physiology*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011506:Proteins; D056465:Retinoblastoma-Binding Protein 1; D050717:Retinoblastoma-Like Protein p130; D016196:S Phase; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D019925:Cyclin A; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050704:E2F4 Transcription Factor; C493763:E2f4 protein, mouse; D010750:Phosphoproteins; D011506:Proteins; C493838:Rbl2 protein, mouse; D056465:Retinoblastoma-Binding Protein 1; D050717:Retinoblastoma-Like Protein p130; D050680:Transcription Factor DP1; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases